Even as the cost and throughput of commercial sequencers has continued to improve over the last 5 years, there is still a need to further reduce sequencing costs, to increase throughput and sequencing accuracy and to reduce the costs associated with sample preparation. Single molecule methods such as the Pacific Biosciences or nanopore technologies have the potential to reduce sample preparation bottlenecks but suffer from very high raw error rates. We are developing the Activator Sequencing technology for single molecule sequencing with low error rates. The method is applicable to a variety of read outs such as fluorescence, luminescence, pH sensing and electrochemistry, many of which can be used in a disposable CMOS chip platform similar to that of Ion Torrent. If successful, Activator Sequencing would enable low-cost, long read length, high accuracy sequencing on a scalable platform capable of leveraging semiconductor industry know-how and investments to yield continued yearly increases in performance based on Moore's Law type decreases in feature size. Activator Sequencing uses a molecular amplifier to convert the products of a single-molecule sequencing reaction into many copies of a readily detectable reporter molecule. Specifically, sequencing-by-synthesis is performed using dNTPs labeled at the terminal phosphate with an enzyme activator. Upon incorporation of a dNTP onto a primed template, an activator is released which can turn an engineered enzyme switch from an off to an on conformation. Each activated enzyme can rapidly generate a multitude of detectable products thereby amplifying the detectable signal from the original dNTP incorporation. For example, while the Ion Torrent system needs many template copies to generate a detectable pH signal, an activator released from a single dNTP molecule can turn on a single enzyme molecule to generate tens of thousands of protons in a few seconds. The generation of multiple copies of a reporter makes it easier to detect nucleotide incorporation thereby allowing single molecule sequencing with low noise. Such single molecule sequencing would simplify sample preparation and enable very long read lengths by eliminating dephasing limitations. If combined with low-cost, highly parallel CMOS sensors, instrumentation costs would be greatly reduced compared to fluorescence instrumentation. Our preliminary results demonstrate that an engineered enzyme switch can function as such a molecular amplifier. The proposed Phase I SBIR grant will demonstrate the ability of Activator Sequencing to use an engineered enzyme switch to perform single molecule sequencing with high accuracy using fluorescence detection. Future work would focus on transferring the technology to a scalable, integrated CMOS sensor.